&lt;b&gt;GLYCOSAMINOGLYCAN METABOLISM BY SCLERODERMA FIBROBLASTS IN &lt;/b&gt;&lt;b&gt;CULTURE &lt;/b&gt;

European Journal of Biochemistry

Kurata

Shinkai

1988

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Collagen synthesis was examined in skin fibroblasts from a patient with a variant of Ehlers-Danlos syndrome. The relative rate of collagen synthesis to total protein synthesis in the patient's fibroblasts was always onehalf of that in fibroblasts from normal controls. Total collagen synthesis, as assessed by quantification of total hydroxyproline, was also significantly lower than that of controls, indicating that the rate of collagen synthesis by the patient's fibroblasts was decreased compared with that by normal fibroblasts. Analysis of procollagen and collagen components showed the absence of the proa2(I) chain and its derivatives. Dot-blot and Northern-blot analyses showed the patient's fibroblasts to contain less than 10% of the mRNAs for proa2(1) found in control fibroblasts. In spite of these results, Southern blot analysis of genomic DNA indicated the presence of the same number of genes for the proa2(I) collagen chain in the patient's fibroblasts as in control fibroblasts, suggesting malfunctioning proa2(I) collagen genes as the cause for failure of the patient's fibroblasts to synthesize proa2(I) collagen chains.Type I collagen is the major structural protein in the body and is found in the skin, bones and tendons in large quantity. It is a trimer composed of two al(1) chains and one a2(I) chain. These chains are synthesized as precursor proal(1) and proa2(I) polypeptides (procollagen chains), which include a central helical collagen domain of about 1000 amino acid residues and amino-terminal and carboxyl-terminal propeptides, together constituting about 500 amino acid residues, that are cleaved after assembly into type I procollagen and secretion from the cells.At least three different groups of inherited connectivetissue disorders have been directly associated with attenuation of type 1 collagen: osteogenesis imperfecta, Ehlers-Danlos syndrome (EDS) and Marfan syndrome [I -51. EDS is characterized by hyperextensibility and fragility of skin, hypermobility of joints and easy bruisability; it has a very heterogeneous etiology and is now subdivided into ten types by clinical and genetic criteria [l -51. Molecular defects of only some types of EDS have been reported, which include Correspondence to R. Hata,

Section: Cell Culturementioning

confidence: 99%

Existence of malfunctioning proα2(I) collagen genes in a patient with a proα2(I)‐chain‐defective variant of Ehlers‐Danlos syndrome

European Journal of Biochemistry

Kurata

Shinkai

1988

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“…Human forearm skin fibroblasts were obtained from three patients with progressive systemic sclerosis (37 -46 years old, PSS1, PSS2, and PSS3) and age-matched normal adults (HSF1, HSF2, HSF3, and HSF4) as described previously [17] and used at population-doubling levels of 10-30. The cells (0.5 -1 x lo5) at similar population-doubling levels were plated onto 35-mm dishes and grown in medium (DMEM-10) for 7-10 days until apparent confluence was obtained.…”

Section: Cell Culturementioning

confidence: 99%

Regulation of collagen metabolism and cell growth by epidermal growth factor and ascorbate in cultured human skin fibroblasts

European Journal of Biochemistry

Sunada

Arai

et al. 1988

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Epidermal growth factor (2 -50 nglml), prepared from mouse submaxillary glands, stimulated growth and the synthesis of non-collagenous proteins and hyaluronic acid, but inhibited collagen synthesis in cultured human skin fibroblasts, both stimulation and inhibition being dose-dependent. All these effects may be intrinsic functions of the epidermal growth factor molecule, because these effects were cancelled by the co-presence of antiserum specific for epidermal growth factor and because they were also observed following the addition of human epidermal growth factor produced urogastrone cDNA. On the other hand, L-ascorbate (vitamin C) stimulated growth and collagen synthesis, as well as synthesis of non-collagenous proteins, with no significant effect on hyaluronic acid synthesis.Co-presence of epidermal growth factor and ascorbate gave additive effects on growth and protein synthesis of the cells. These results suggest that the two growth-promoting factors, epidermal growth factor and L-ascorbate, modulate metabolism of extracellular matrix components as well as cell growth in a quite different manner in human skin fibroblasts.Epidermal growth factor (EGF), first isolated from male mouse submaxillary glands, is a 53-amino-acid polypeptide [l]. It modulates the growth properties of many types of cells including cells of mesenchymal origin such as fibroblasts [2] and osteoblasts [3, 41 in addition to cells of epithelial origin [l]. EGF also regulates a variety of cellular functions including metabolism of extracellular matrix components [4 -71. Urogastrone, isolated from human urine, was noticed by its inhibition of gastric acid secretion and was found to correspond to human EGF (h-EGF) [8, 91.L-Ascorbate (vitamin C), which was first found by its antiscorbutic activity, also has been shown to have multifarious functions on various kinds of cells [lo, 111. One of the most important functions of ascorbate may be its activity as a cofactor for the hydroxylation of proline and lysine residues in collagen [12], which is the most abundant protein present in the animal body. It is also reported that ascorbate increases messenger RNA levels for collagen in cultured fibroblasts [13]. In the course of an investigation on regulatory mechanisms involved in the metabolism of extracellular matrix compoCorrespondence to R. Hata,

“…Fibroblasts were obtained from rat abdominal skin and cultured in Dulbecco's modified Eagle's medium supple. mented with 10% bovine fetal serum as described previously (Ninomiya et al, 1982). Metabolic labeling of the cells.…”

mentioning

confidence: 99%

Activation of collagen synthesis in primary culture of rat liver parenchymal cells (hepatocytes)

Journal Cellular Physiology

Ninomiya

Sano

et al. 1985

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An increase in collagen synthesis by hepatic parenchymal cells (hepatocytes) was observed during 8 days in primary culture by the quantification of total [3H]hydroxyproline as a marker of total collagen synthesis and the ratio of [3H]hydroxyproline in the high-molecular-weight fraction to total [3H]hydroxyproline as a marker of collagen degradation after incubation of the cells with [3H]proline for 24 h. Type analysis of the collagen produced by the cells after 8 days in culture showed the presence of type I and type III collagens in addition to the components corresponding to type IV and type V (alpha A and alpha B) collagens. Only the latter two types were found in the collagens produced by the cells after 2 days in primary culture. The purity of the hepatocytes inoculated was 97%, and the majority of the contaminating small cells were erythrocytes. The rate of serum albumin synthesis, which is a typical function of the hepatocytes, was constant or increased during the culture period. Immuno-electron microscopic observation indicated the production of type I collagen by the hepatocytes after 8 days in primary culture. These results are explained only by the activation of collagen synthesis in the day-8 hepatocytes in primary culture.